We demonstrate that this isotropic opto-magnetic effect, which can be called inverse magneto-refraction, is allowed in a material of any symmetry. Its existence is corroborated by the experimental observation of terahertz emission by spin resonances optically excited in a broad class of iron oxides with a canted spin configuration. From its strength we estimate that a sub-picosecond modification of the exchange interaction by laser pulses with fluence of about 1 mJ cm−2 acts as a pulsed effective magnetic field of 0.01 Tesla.Ultrafast non-thermal manipulation of magnetization by light relies on either indirect coupling of the electric field component of the light with spins via spin-orbit interaction or direct coupling between the magnetic field component and spins. Here we propose a scenario for coupling between the electric field of light and spins via optical modification of the exchange interaction, one of the strongest quantum effects with strength of 10 In the ultrafast manipulation of magnetization by light, it is thought that the electric field couples only indirectly to spins via spin-orbit interaction. Here, the authors demonstrate inverse magneto-refraction, a direct optical modification of the exchange interaction allowed for any material symmetry. J is the exchange integral; ith and jth adjacent magnetic ions. The antisymmetric part D and called Dzyaloshinskii–Moriya interaction, gives rise to canted antiferromagnetism2The symmetric part of the exchange interaction between spins is responsible for the very existence of magnetic orderingJ from the demagnetization dynamics. Nevertheless, a direct, truly ultrafast effect of the electric field of light on the exchange interaction must be feasible in any material. In a medium of arbitrary symmetry, such an effect may be expressed phenomenologically by introducing an isotropic term in the Hamiltonian of the two-photon interaction between the light and spinsThe ability to control the exchange interaction by light has intrigued researchers in many areas of physics, ranging from quantum computing4578109Iopt is the intensity of light; α and β are some scalar and vector coefficients, respectively. The presence of the interaction Hamiltonian (1) manifests itself as a magnetic refraction, described by an isotropic contribution to the dielectric permittivity ɛIMR∼M2 that leads to a dependence of the refractive index on the magnitude of the magnetization M15J=αIopt, to the symmetric Heisenberg exchange integral J, whereas the second term describes the intensity dependent contribution, ΔD=βIopt, to the Dzyaloshinskii–Moriya vector D. Recently the effect of isotropic magneto-refraction has been used to probe d–f exchange in EuTeTi acting on a spin Si due to the light-induced perturbation of the exchange parameterswhere where γ is the absolute value of the gyromagnetic ratio. The torque (2) is zero in materials with collinear magnetic configurations since In a broad class of transition metal oxides the magnetic order is governed by indirect exchange via ligand ions (superexchange)3, rare-earth orthoferrites RFeO3 (R stands for a rare-earth element) and hematite α-Fe2O3, are natural candidates for observing such ultrafast optical modification of the superexchange interactions. In these compounds the Fe3+ ions  form two magnetic sublattices, the spins of which are antiferromagnetically coupledD/J between the antisymmetric and symmetric exchange parameters. Thus, one could expect that an ultrafast optical perturbation of the exchange parameters could also change the ratio D/J and thereby trigger, by the torque defined in 2728303132Antiferromagnetic iron oxides possessing weak ferromagnetism, such as iron borate FeBOHere we reveal the inverse magneto-refractive effect to be responsible for ultrafast modulation of the superexchange interaction in a very broad class of canted antiferromagnets. Our findings are supported by a low-energy theory for the magnetic interactions between non-equilibrium electrons subjected to an external time-dependent electric field. We present quantitative estimates of the strength and timescale of the optical perturbation of the exchange parameters.3 and ErFeO3 which revealed the optical excitation of the high-frequency quasi-antiferromagnetic mode in these compounds along with the low-frequency quasi-ferromagnetic mode, another form of the antiferromagnetic resonance in canted antiferromagnets which involves the precession of the magnetization with no change in its lengthD/J via inverse magneto-refractive effect, in particular.Recently we reported measurements of THz emission signals in the rare-earth orthoferritesD/J, we have studied the THz emission from a single FeBO3 cut perpendicularly to the z-crystallographic axis so that it lacks significant in-plane anisotropy of both optical and magnetic properties. The magnetization lying in the plane of the sample was aligned horizontally by a constant bias magnetic field of ∼0.1 T. The sample was illuminated by ∼100-fs laser pulses with their photon energy centred at 1.55 eV. We performed time-resolved detection of the THz radiation emitted from the sample in the direction of the z axis  plane at the frequency of the quasi-ferromagnetic mode (∼100 GHz)38z-cut TmFeO3 sample, with a net magnetic moment oriented upwards, a laser-induced spin-reorientation transition should trigger the quasi-ferromagnetic mode in such a way that the Mx component of the magnetization decreases. The observed difference in the phases between the two oscillations shows that the quasi-antiferromagnetic mode is triggered in such a way that the Mx component increases, which means that the canting angle becomes larger. Such a behaviour can only be explained by assuming that the quasi-antiferromagnetic oscillations are triggered by an increase of the ratio of the exchange parameters D/J. If this conclusion is true, in the x-cut sample the initial phases of the two modes must be the same, since the spin reorientation in this sample proceeds in the opposite direction. Measurements in the vicinity of the spin-reorientation temperature in ErFeO3 cut perpendicular to the x axis confirm this conclusion , the x axis  and the y axis, . The iron borate FeBO3 sample (370-μm thick) and haematite α-Fe2O3 sample (500-μm thick) were cut perpendicularly to the z axis. The lateral size of all plates was ∼5 mm.The crystals used in the present study were grown by floating zone melting (orthoferrites) and from the gas phase (iron borate and hematite) The orthoferrite samples were 60–100-μm thick and cut perpendicularly to the −2. The electric field of the emitted THz wave was measured by the electro-optical sampling technique. The sample was held inside a closed cycle, helium cryostat .A conventional time-domain THz spectrometer was used in the measurements. The THz spectrometer was powered by a Ti:sapphire amplified laser, emitting a sequence of optical pulses  with the repetition frequency of 1 kHz. Each laser pulse was divided into a stronger pump pulse and a weaker probe pulse. The pump spot size was larger than the aperture in the sample holder (∼2 mm in diameter) to provide a quasi-uniform excitation with a fluence of ∼1 mJ cmWe use a general formalism in which magnetic interactions are obtained from a purely electronic model by introducing small time-dependent rotations of the spin quantization axes as was recently described in ref. . For thed orbital and atom 1 contributes one filled (oxygen) p orbital. The Hamiltonian consists of a local part Hloc and a time-dependent hopping term H′(t):This method is implemented for the simplest model system that exhibits the physics of superexchange, which consists of a chain of three atoms, labelled as 0, 1 and 2. Atoms 0 and 2 correspond to transition metal sites with one partially filled σ={↑,↓} at site j, and ɛd and ɛp are the orbital energies of d and p orbitals, respectively, and U is the local (Hubbard) interaction energy associated with d orbitals. H′(t) accounts for hopping between p and d orbitals; t0 is the equilibrium hopping parameter, while ϕ(t) is the time-dependent Peierls phase, which absorbs the effect of the time-dependent electric field. In the Coulomb gauge and for a spatially uniform vector potential the Peierls phase is given byHere A||(t) is the component of the vector potential parallel to the chain and a is the lattice spacing. In the chosen gauge, the electric field is related to the vector potential as U, U+ɛd−ɛp>>t0 and a total filling of four electrons. To compute the non-equilibrium functions, this cluster model is solved numerically using exact diagonalization of the time-dependent Schrödinger equation. The electric field is taken as the product of an oscillatory component and a gradually changing envelope function with a rising time in the order of ∼10 periods of oscillation. The frequency ω of the oscillating part is below the charge-transfer gap, which prevents direct charge-transfer transitions, consistently with the experimental conditions.where Further details related to the theoretical and numerical methods are discussed in How to cite this article: Mikhaylovskiy, R. V. et al. Ultrafast optical modification of exchange interactions in iron oxides. Nat. Commun. 6:8190 doi: 10.1038/ncomms9190 (2015).Supplementary Figures 1-9, Supplementary Notes 1-6 and Supplementary References


Given an uncharacterized compound, can we develop a computational method to fast identify which ATC class or classes it belongs to? The information thus obtained will timely help adjusting our focus and selection, significantly speeding up the drug development process. But this problem is by no means an easy one since some drug compounds may belong to two or more than two ATC classes. To address this problem, using the DO (Drug Ontology) approach based on the ChEBI  database, we developed a predictor called iATC-mDO. Subsequently, hybridizing it with an existing drug ATC classifier, we constructed a predictor called iATC-mHyb. It has been demonstrated by the rigorous cross-validation and from five different measuring angles that iATC-mHyb is remarkably superior to the best existing predictor in identifying the ATC classes for drug compounds. To convenience most experimental scientists, a user-friendly web-server for iATC-mHyd has been established at  Open the web-server at http://www.jci-bioinfo.cn/iATC-mHyb, the top page of iATC-mHyb will appear on the computer screen, as shown in Figure Step 2. Either type or copy/paste the formulae of query compounds into the input box at the center of Figure Step 3. Click on the Submit button to see the predicted result. For example, if using the formulae of the five compounds in the Example window as the input, one will see Figure 1) Compound-1 belongs to three different ATC-classes; i.e., classes 3, 5 and 9, which are predicted by iATC-mDO subpredictor, meaning that the compound is covered by the ChEBI database. (2) Compound-2 belongs to only one ATC-class; i.e., class 3, which is predicted by iATC-mDO subpredictor, meaning the compound is covered by the ChEBI database. (3) Compound-3 belongs to four different ATC-classes; i.e., classes 3, 4, 10 and 12, which are predicted by iATC-mDO subpredictor, meaning that the compound is covered by the ChEBI database. (4) Compound-4 belongs to three different ATC-classes; i.e., classes 4, 5 and 13, which are predicted by iATC-mISF subpredictor, meaning that the compound is not covered by the ChEBI database. (5) Compound-5 belongs to two different ATC-classes; i.e., classes 4 and 12, which are predicted by iATC-mISF subpredictor, meaning that the compound is also not covered by the ChEBI database. All these results are fully consistent with the experimental observations.Step 4. Click on the Citation button to find the key relevant papers that have been used to document the detailed development and algorithm of iATC-mHyb.Step 5. Click the Supporting Information button to download the all the “Supporting Information” files mentioned in this paper.1) how to construct or select a valid benchmark dataset to train and test the predictor; (2) how to formulate the drug compound samples with an effective mathematical expression that can truly reflect their essential correlation with the target concerned; (3) how to introduce or develop a powerful algorithm (or engine) to run the prediction; (4) how to properly conduct cross-validation tests to objectively evaluate the anticipated accuracy; (5) how to provide a web-server and user guide to make users very easily to get their desired results. Below, let us to address these point-by-point.As demonstrated in a series of recent method-developing studies , 60–65, For facilitating comparison, in this study we used the same benchmark dataset (Supporting Information S1) as used in , 4. It cm-th ATC class , and ∪ denotes the symbol for “union” in the set theory. Listed in Table where the subset ss m = 1,,3,...,14As we can see from the table, among the 3,883 drugs, 3,295 occur in one class, 370 in two classes, 110 in three classes, 37 in four classes, 27 in five classes, 44 in six classes, and none occurs in more than six classes. For such a multi-label system, let us use a more intuitive method to describe the benchmark dataset as given in Supporting Information S2, where the symbol “1” under the title of “ATC classification” means the drug concerned occurs in the corresponding class, “0” means not.Eq.1 is defined byOne of the keys in developing a powerful predictor is to formulate the samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the target to be predicted . In the T is the transposition operator, α1 stands for its maximum DO similarity score with the drugs in the subset 2 for its maximum DO similarity score with the drugs in the subset 3 for that in subset where lculated , 68 fromlculated  via KEGGlculated .ftp://ftp.ebi.ac.uk/pub/databases/chebi/ontology/), and can be defined by Eq.Note that, of the 3,833 drug compounds in the benchmark dataset, only 1,144 can be found in the current ChEBI database  classifier has been adopted to predict the ATC-classes, as described below.i-th drug in the benchmark dataset Suppose the Li given byAnd its attribution in a multi-label system can be formulated as a vector whereLikewise, for a query drug or compound, we haveIts attribution label vector in the ACT system is predicted aswherem in Eq.8 is given byThe ΔDq – Di||2 is the Euclidean distance in the 14-D space  to predict the protein subcellular localization , 70, enzwhere “Hyb” means “hybridization” with the iATC-mISF predictor .One of the important procedures  in develThe metrics used to measure the prediction quality for multi-label systems are much more complicated than those for single-label systems. To make them more intuitive and easier to understand for most experimental scientists, the following five metrics were introduced by Chou : (1) “aiThe aforementioned Chou's five metrics can be formulated as {Aiming=N is the total number of the samples concerned, M is the total number of labels for the investigated system, k-th sample, k-th sample, andWhere The above approach had been effectively used to study various multi-label systems, such as those in which a protein may occur in two or more different subcellular locations –23, 76, Three cross-validation methods are often used in statistical prediction. They are: (1) independent dataset test, (2) subsampling  test, and (3) jackknife test . Of thesth sub-equation in Eq.Since Eq.http://www.jci-bioinfo.cn/iATC-mHyb along with a step-by-step guide. Moreover, the MATLAB code for the new method is also available as in Supporting Information S4, which can be directly downloaded from the web-server.A new method for predicting the ATC classes has been developed by hybridizing the drug ontology approach with the best existing ATC predictor. The new predictor has outperformed the best existing ATC predictor in all the five metrics used to examine the prediction quality of a predictor for multi-label systems, particularly in the “absolute true” rate and the “absolute false” rate, the two most difficult-to-improve indexes. To maximize the users’ convenience, a publically accessible web-server has been established at


Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.While the association of Epstein–Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity  Typical of c-MYC-driven lymphomas, BLs proliferate rapidly but are also sensitive to apoptosis under conditions of stress.2Epstein–Barr virus (EBV)-positive Burkitt lymphoma (BL), an aggressive and difficult to treat malignancy, is endemic (eBL) in sub-Saharan Africa, where it accounts for around half of all childhood lymphomas. BL also occurs worldwide at lower incidence, and in these cases, known as sporadic BLs (spBL), EBV is found in 15–85% of tumours, varying by geographical region.et al.3). It was shown using the Eμ-Myc transgenic mouse model of human BL  that blocking apoptosis through enforced expression of BCL-2 prosurvival proteins or deletion of BH3-only proteins or BAX greatly accelerates lymphoma development.7Cellular proapoptotic BH3-only proteins  induce cell death by unleashing the proapoptotic multi-BH domain executioner BCL-2 family members, BAK and BAX, allowing them to form pores in the mitochondrial outer membrane, which commits the cells to apoptosis. BH3-only proteins can achieve this by binding and inhibiting prosurvival BCL-2 family proteins , which in the absence of an apoptotic stimulus restrain BAK and BAX, thereby preventing cellular destruction. Additionally, BIM, PUMA and tBID can directly activate BAX and BAK , two non-coding RNAs (EBER1 and EBER2), and two families of microRNAs (BART microRNAs (miR-BARTs) and miR-BHRF1s), which together drive proliferation and promote cell survival   .10 Cell lines derived from these tumours show marked resistance to apoptosis due to epigenetic silencing of the BIM promoter11 and functional inhibition of BIM, PUMA, BID and BAK by the viral BCL-2 homologue, BHRF1.12Crucially, however, most of the Latency III genes are not expressed in established BLs. Instead, in BLs, EBV exhibits more restricted forms of latency characterised by expression of EBNA1, the EBER transcripts and the miR-BARTs . Only a 14 and precise Ig-MYC chromosomal translocations.16 It is therefore unsatisfactory to introduce the virus or viral genes into EBV-negative spBL lines to study the role of EBV in eBL. Instead, efforts have focused on trying to rid EBV-positive eBLs of the virus to assess the contribution of EBV to the growth and survival of BL in an isogenic system. Treatment of EBV-positive BL cells with a dominant-negative form of EBNA1 leads to loss of EBV genomes and widespread apoptosis.20 While implying that EBV is essential for the continued survival of BL cells, this method yielded few EBV-loss clones for mechanistic studies. Hydroxyurea treatment can also eradicate EBV, but these BL clones do not show a consistent apoptosis predisposition phenotype.21 Additionally, an unusual EBV-positive spBL (Akata-BL) cell line has been reported to spontaneously lose EBV in vitro, yielding clones with impaired cell growth.22EBV-positive and -negative BLs are genetically distinct, differing in terms of their cellular mutational profiles24 The present study resolves the unmet need for a systematic approach to analyse multiple EBV-loss BL clones on several tumour backgrounds both in vitro and in vivo in a xenograft model of BL. This work has shown unequivocally that EBV in a Latency I infection can protect BL cells from apoptosis mediated by the proapoptotic BH3-only proteins, BIM and PUMA.A significant limitation of these previous studies has been the small number of tumour backgrounds and/or clones analysed. BL, like many cancers, can exhibit considerable inter- and intratumoral genetic heterogeneity.A panel of EBV-positive BL cell lines (1 spBL and 11 eBLs) were seeded at single-cell dilutions to establish more than 1800 clones. These clones were screened for EBV episome copy number by quantitative, real-time PCR (q-PCR); the results are summarised in Before investigating the phenotype of the EBV-positive clones, their EBV infection status was fully characterised. This included flow cytometry for EBERs to confirm that all cells carried the virus, as well as immunoblotting and q-PCR for 45 EBV transcripts to ensure that the clones retained Latency I gene expression  .in vivo, isogenic EBV-positive and EBV-loss clones derived from three eBL backgrounds , were transplanted by intraperitoneal injection into female NSG mice and the animals monitored for tumour burden. Tumours arose in the spleen, ovaries and pancreas and resembled human BL in terms of pathology and histological ‘starry sky’ appearance . To broaden these assorted but limited analyses, each of the Latency I-associated genes were introduced into multiple EBV-loss clones derived from several tumours. 34 were used to express EBNA1 protein and the BART miRs in EBV-loss cells at levels equivalent to those detected in EBV-positive BLs (EcoRI J region of the Akata strain EBV genome (KC207813.1),29 was used to efficiently express physiologically high levels of EBER RNAs , is highly contradictorytive BLs . A derivBER RNAs .35 Expression of cluster 1 miR-BARTs, cluster 2 miR-BARTs or miR-BART-5 alone also conferred no protection to EBV-loss BL clones 40 and a variant of the B95.8 prototype strain termed ΔCpWp-B95.8 (B-V), which is forced to adopt a Latency I infection because of genomic deletions spanning the Latency III-associated viral promoters, Cp/Wp41 EBV. These experiments are not trivial as reinfection with EBV usually gives rise to the extensive Latency III infection rather than a restricted Latency I pattern, Cp/Wp41 . ReinfecBamHI A genomic locus, which is naturally deleted in B95.8 and its derivative, B-V.43 These data highlight the clonal variation and emphasises the need to analyse multiple clones before drawing general conclusions.Importantly, Ak-V could protect EBV-loss BL cells from apoptotic stimuli; six cell lines were fully protected, two were substantially protected and one (Akata n2) showed no protection . Reinfec46To determine which cellular genes are modulated by EBV in Latency I BL, RNA was extracted from EBV-positive and -loss clones from four BL backgrounds  and microarray gene expression analysis was undertaken. Surprisingly, no genes were significantly changed more than twofold in EBV-loss compared with EBV-positive clones across all tumours. Additionally, no differences in steady-state expression of BCL-2 family transcripts were observed . We wereversus 79% viability), with concordant cleavage of PARP and increased caspase activation , could be useful clinically in the treatment of EBV-positive BLs.To our knowledge, this is the most extensive analysis of the role of EBV in BL. The large-scale, single-cell cloning of BL cell lines and isolation of only 61 spontaneous EBV-loss clones from >1800 clones strongly indicate that EBV is highly selected for in BL. Consistent with reports that EBV’s role in BL is to protect cells from death stimuli,43 but also a number of transcripts of unknown function that appear to be highly transcribed in latent BL cells.53 Therefore, it is likely that genes encoded within the B95.8 deletion contribute to Latency I-mediated protection from apoptosis. Further development of rEBV genomes is required to decode the precise contribution of these complex transcripts to BL.Importantly, reinfection of EBV-loss clones with recombinant Ak-V could fully restore apoptosis resistance, confirming that EBV is directly responsible for the increased resistance to cell death. Interestingly, B95.8-derived rEBV, which harbours a large genomic deletion, could partially, but never fully restore apoptosis protection to BL cells. This genomic deletion spans not only a number of the miR-BARTs31 EBER RNAs were shown to confer tumorigenicity to EBV-loss Akata-BL in vivo and to enhance the growth properties of cells in vitro, in part through upregulation of BCL-2 and IL-10.54 Yet we found no evidence that BCL-2 or IL-10 were more highly expressed in EBER-expressing or EBV-positive cells compared with EBV-loss cells. Consistent with our findings, others reported that expression of EBERs alone did not restore protection from apoptosis in Akata-BL cells in vitro, but that coexpression of EBNA1 and EBERs rendered two EBV-loss Akata-BL clones more tumorigenic in vivo.25 The authors attributed this to increased EBER expression in the EBNA1-/EBER-positive EBV-loss cells. However, an alternative interpretation is that EBNA1 enhances the tumorigenicity of EBER-expressing cells by functional cooperation. Another report attributed apoptosis protection to the miR-BARTs,18 but the considerable clonal variation (between cell lines and viruses) in this study precludes firm conclusions. BART miRs have been reported to downregulate PUMA in epithelial cells36 (a finding we could recapitulate in 293 cells), but consistent with other groups,56 this downregulation was not apparent in B-lineage-derived cell lines.No Latency I-encoded gene product alone  could restore apoptosis resistance to EBV-loss BLs. This indicates a requirement for cooperation between multiple genes, including transcripts from the B95.8 deletion. There is much debate in the literature in this regard. EBNA1 is essential for virus genome maintenance and also reported to function in cell survival in certain circumstances.3 In addition to the upregulation of BIM and PUMA proteins after apoptosis induction in EBV-loss cells, increased expression of BIM and PUMA transcripts was also observed compared with EBV-positive cells, although this did not reach statistical significance. Consistent with cooperation between multiple EBV genes, we therefore hypothesise that the repression of BIM and PUMA by EBV occurs at both the protein and RNA levels and involves multiple molecular mechanisms.Our work implicates the BH3-only proteins BIM and PUMA as the key cellular genes regulated by EBV in Latency I BL. These proteins are critical initiators of the intrinsic apoptotic pathway, able to bind and inhibit all BCL-2 prosurvival proteins and also capable of directly activating the executioners of apoptosis, BAX and BAK.56 and therefore likely inhibit their translation. Additionally, EBERs have been shown to activate AKT/PI3K signalling,57 which is important for BL cell survival.58 Interestingly, tonic AKT/PI3K signalling has been reported to suppress induction of PUMA and BIM, by both transcriptional and post-translational mechanisms, and consequently suppress apoptosis in leukaemia cells following growth factor withdrawal.59 Furthermore, EBNA1 is able to deplete cells of SMAD2,61 which can lead to inhibition of PUMA expression through the TGF-β pathway.63 A recent study showed that both RNA and DNA viruses, typified by Semliki Forest virus and herpes simplex 1 virus, respectively, control intrinsic apoptosis via regulation of PUMA.64 Therefore, it is likely that Latency I EBV genes co-operate to regulate PUMA and BIM both directly and indirectly and that multilateral regulation of prodeath BH3s is critical and conserved in human viruses.BART miRs are predicted to bind multiple sites in the 3′-UTRs of BIM and PUMA65 Therefore, EBV infection of a preneoplastic cell bearing a c-MYC chromosomal translocation would be predicted to accelerate tumour development by blocking BIM and PUMA upregulation. Our work opens up the possibility to incorporate BH3-mimetic drugs  into the treatment regimen for EBV-positive BL to help improve patient survival, which remains woeful in certain patient groups.The blocking of BIM and PUMA upregulation by EBV has important implications for BL pathogenesis as it has been shown that deregulated c-MYC expression can sensitise cells to apoptosis and that this predisposition must be overcome for BL to develop.9 When referring to BL-derived subclones, the prefix ‘P’ indicates an EBV-positive clone, whereas ‘n’ denotes a clone that has lost the virus. BL cell lines were maintained in RPMI-1640 supplemented with 10% FCS , 6 mM glutamine, 1 mM pyruvate, 50 μM α-thioglycerol, 20 nM bathocupronine disulphonic acid and 8 μg/ml gentamycin . All cells were routinely grown at 37 °C in a humidified atmosphere containing 5% CO2.Akata-BL was a kind gift from Prof. Kenzo Takada, Rael-BL and the lymphoblastoid cell line (LCL), Raji-BL, X50-7, were kind gifts from Prof. George Klein. All other BL cell lines were established by Prof. Alan Rickinson and co-workers. Jurkat clone E6.1 was from the European Collection of Cell Cultures, HEK-293 cells were obtained from the American Tissue Culture Collection (product number: ATCC CRL-1573) and 293FT cells (R700-07) were from Life Technologies . Clonal cell lines were established by single-cell cloning carried out as described previously.4/cm2 in the presence of inducers or inhibitors of apoptosis, including: ionomycin ; etoposide ; Fas-activating CH11 antibodies  ; roscovitine ; staurosporine  , Danvers, MA, USA); IgM crosslinking antibodies (5 μg/ml) ; the human cytokine, IL-10 (50–500 ng/ml) ; Q-VD.OPh (20 μM)  and/or a vehicle-only control (DMSO) as stated, unless otherwise specified in figure legends. Where inducible lentiviruses were used, gene of interest expression was induced by the addition of 1 μg/ml doxycycline 24 h before the experiment set-up. All assays were carried out in triplicate and on at least three occasions. Data are expressed as mean and standard deviation of independent triplicates unless otherwise specified. Cell viability was determined by dual staining of cells with Annexin-V-APC and PI or PI staining alone and analysed by flow cytometry. Surface CD95 staining was determined using the DX2-PE-CY7  as per the manufacturer’s instructions. A minimum of 10 000 events was recorded for all samples and data were analysed using the FlowJo Software  or Accuri C6 Software .BL cells were seeded at 9x10β-actin ; caspase-3, -7 and -9 ; DEDD2/FLAME3 ; PUMA ; BIM ; CFLAR ; CASP8AP2 ; BID ; BAD ; BCL-2 ); BCL-X ; MCL-1 ; cIAP1 , Livin ; Survivin ; XIAP ; NOXA ; BAK ; and BAX . Protein size analysis and densitometry were carried out using the Chemidoc MP system equipped with the ImageLab v.5.2 Software . Size markers were SeeBlue Plus 2 (Life Technologies) and densitometry calculations were normalised to endogenous control proteins, calregulin or β-actin.Proteins in whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes that were probed using antibodies raised against: EBNA1 (AMo serum); EBNA2 (PE2); LMP1 (CS1-4); BHRF1 (5B11); Cg-Prkdcscidil2rgtm1Wjl/SzJ) mice were given intraperitoneal or subcutaneous injections of BL cells diluted in PBS. Cell numbers used were 1 × 106 for Kem-BL, Awia-BL and Mutu-BL clones or 1x107 for Akata-BL. Mice were monitored for signs of tumour growth and killed when their tumour burden reached 1 cm3. Lymphoma cell suspensions made from explanted tumour tissues were shown to be >99% positive for human CD19 FACS  to confirm that tumours were derived from xenotransplanted BL cells.Mice were kept in specified pathogen-fee animal areas at the Walter and Eliza Hall Institute of Medical Research (WEHI). All experiments involving mice were conducted in accordance with the requirements of the WEHI Animal Ethics Committee. Eight-week-old, female NSG  in the presence of 32P-labelled dCTP . Probe hybridisation and washes were carried out using the XpressHyb System  according to the manufacturer’s instructions. Exposures were carried out at −80 °C for 2–14 days.RNA was isolated using Trizol reagent, separated on precast urea gels (both Life Technologies) and blotted onto nylon membranes. Radio-labelled DNA probes were generated by PCR amplification of the ACTB and GAPDH. Absolute quantitation of EBV transcripts and housekeeping controls were quantitated by real-time PCR relative to a DNA plasmid standard to enable absolute copy numbers to be calculated. This method and all the relevant primer-probe sets have been described elsewhere.53 Stem-loop primed cDNA was prepared with miR-specific primers and TaqMan microRNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s guidelines and miR expression was quantified using commercially available TaqMan primer/probe mixes (Life Technologies). EBV genome load was calculated by DNA q-PCR. Briefly, DNA was extracted using the GenElute DNA Kit (Sigma) according to the manufacturer’s instructions and 5 ng DNA was used per q-PCR assay. Genome load was calculated by quantitating copies of the EBV polymerase gene (BALF5) normalised to β-2-microglobulin (β2M), assuming two genomic copies of β2M per cell. Each measurement was made in triplicate.Total RNA was prepared using miRVana RNA Extraction Kit (Life Technologies) according to the manufacturer’s instructions. Cellular apoptosis-related gene expression was quantified using TaqMan Low Density Array human apoptosis panel cards . Random hexamer-primed cDNA was prepared using the qScript cDNA Supermix . All experiments were carried out in triplicate and samples were run in triplicate. Data were analysed using Data Assist v.3.01 (Life Technologies) using the ΔΔCt method, and then normalised to the mean of two endogenous controls, S-variant proteins were expressed from an F-UTG-derived lentivirus, which constitutively expresses GFP and drives expression of the gene of interest from a dox-inducible, polII, Trex promoter, as described previously.67 Recombinant Akata virus (Ak-V)-producing cells were a kind gift from Prof. Kenzo Takada and the CpWp-KO B95.8 virus (B-V) was developed by Dr. Rosemary Tierney. Detailed descriptions of all recombinant EBVs and expression constructs are available in The EBNA1 protein, clusters of BART miRs and BIM68 or from Ak-V-infected BL cells by crosslinking surface IgG40 as described previously. Before EBV infection, EBV-loss BL clones were transduced with an expression construct expressing the EBV receptor, human CD21, to enhance the infection efficiency.69Lentivirus containing supernatants were produced by transfecting 293FT cells (Life Technologies) with the expression constructs of interest alongside the envelope plasmid, pMD2.G, and the packaging plasmid, psPax2, using Lipofectamine 2000 (Life Technologies). Infectious virus stocks produced from B-V BAC-carrying 293 clones by BZLF1/BALF4 transfections-BH3 variant functional assays, reinfection studies, q-PCR and immunoblot gene expression analyses the significance of differences between cell lines were calculated using an unpaired, two-tailed, Student’s T-test. For apoptosis assays to characterise the effect of EBV loss, unpaired, two-tailed Student’s T-tests were carried out on clones from each background. Additionally, the difference in cell survival between all EBV-positive clones and all EBV-loss clones across different BL backgrounds was analysed by two-way ANOVA. Survival comparisons of mouse cohorts were calculated using log-rank (Mantel–Cox) analysis. T-tests, Mantel–Cox and ANOVA analyses were carried out in the GraphPad Prism 5.0 Software . Differences were considered significant where the P-value fell below 0.05 and were classified as follows: *P<0.05, **P<0.01, ***P<0.001 and nsP>0.05 (not significant).For BIM


Despite anti-VEGF therapy, some patients develop chronic diabetic macular edema. The objective of this study was to evaluate anatomic and visual outcomes of switching patients with chronic DME from intravitreal bevacizumab or ranibizumab to intravitreal aflibercept injection.In this retrospective observational case series, 11 eyes with recalcitrant diabetic macular edema (DME) were evaluated 6 months prior to and 6 months following initial intravitreal aflibercept injection (IAI). Recalcitrant DME was defined as having a thickened retina (≥350 μm) on spectral domain optical coherence tomography (SD-OCT) with persistent cystic changes  over 6 months prior to intravitreal aflibercept switch despite aggressive treatment for DME during this time.One hundred and forty-seven patients in total were treated with IAI during this time, and of these, 31 patients were treated with IAI for DME. 18 eyes had less than 4 treatments within the 6 months prior to switch to IAI, 6 patients had a central retinal thickness (CRT) on SD-OCT of less than 350 μm at time of switch to IAI, and 2 patients had a greater than 15% decline in CRT on SD-OCT over the 6 months prior to switch to IAI. A total of 11 patients were included in the study. Over the 6 months prior to switch, the mean change in central retinal thickness was +18.6% and over the 6 months following switch to aflibercept the mean change in central retinal thickness was −27.1%. Switching to a regimen of at least 3 intravitreal aflibercept injections over 6 months resulted in some anatomic improvement and improvement or stabilization of Snellen visual acuity in all eligible patients.In patients with recalcitrant diabetic macular edema, switching to intravitreal aflibercept resulted in improved a 25% or more decrease in central retinal thickness in 81% (9/11) patients at 6-month follow-up. Sixty-three percent (7/11) had improvement in Snellen visual acuity after switching to intravitreal aflibercept injection, suggesting some reversibility of functional damage. Diabetic macular edema (DME) is the leading cause of central vision loss in people with diabetes and is characterized by edema and thickening of the macula . From thIntravitreal ranibizumab  became the first VEGF inhibitor FDA-approved for the treatment of DME in 2012 following the RIDE and RISE trials, which showed significant superiority of both 0.3 and 0.5 mg ranibizumab groups over sham injection in improving visual acuity and decreasing central retinal thickness in patients with DME , BOLT 77, and a In July 2014, the FDA approved intravitreal aflibercept , a highly specific fusion protein with high VEGF-binding affinity, for the treatment of DME. This approval followed the VIVID and VISTA clinical trials, which showed superiority in anatomic and visual outcomes in patients treated with intravitreal aflibercept in comparison to laser treated controls. Patients in these studies required a 3-month washout period for patients previously treated with anti-VEGF agents .In March 2015, Protocol T of the Diabetic Retinopathy Clinical Research Network (DRCR.net) published results of a study comparing the relative efficacy of intravitreal bevacizumab, ranibizumab, and aflibercept in treatment of DME. They found that all three anti-VEGF treatments improved vision in center-involving DME, with the relative effect depending on baseline vision. In cases of mild vision loss (20/40 or better), there was no significant difference between the three treatment groups. However, in cases with poorer baseline visual acuity (20/50 or worse), aflibercept was significantly more effective than both ranibizumab and bevacizumab in improving vision. Although Protocol T provides data on the comparative effectiveness across the 3 anti-VEGF agents, this study was not designed to inform on the relative efficacy when switching agents, and excluded eyes that received an anti-VEGF injection within the preceding 12 months [Since FDA approval of aflibercept for the treatment of DME and results from Protocol T, many ophthalmologists are switching patients from ranibizumab or bevacizumab to aflibercept, especially those with minimal or incomplete response to the former treatments. In the current study, we evaluated the outcome of switching to aflibercept (IAI) in patients with DME considered to be “recalcitrant” to therapy. Patients were deemed recalcitrant if they had minimal decrease in central retinal thickness (CRT) on spectral domain optical coherence tomography (SD-OCT) despite aggressive treatment during the prior 6 months.A retrospective, observational case series was performed to study the outcome of switching to intravitreal aflibercept in eleven eyes with DME who were considered to be sub-optimal responders to standard of care and were considered to be “recalcitrant” to current therapy. The Institutional Review Board at Tufts Medical Center approved the study protocol for human subjects. The study was complaint with the Health Insurance Portability and Accountability Act of 1996 and adhered to the tenets of the Declaration of Helsinki.Intravitreal injection logs for all patients treated with intravitreal aflibercept 2.0 mg (0.05 mL) at two clinical sites of the Vitreoretinal Service at Tufts Medical Center Department between March 2014 and July 2015 were reviewed. Electronic medical records were then reviewed to identify possible participants that were treated with intravitreal aflibercept injection for DME.Inclusion criteria included ability to provide written informed consent, age 18 years or older, adequately clear media for SD-OCT imaging, and Snellen visual acuity of 20/40 to 20/300 in the study eye. Anatomic criteria for inclusion required CRT ≥ 350 μm on SD-OCT and recalcitrant DME was defined as persistent cystic change with ≤15% decrease in CRT over the 6 months prior to IAI switch despite having at least 4 total treatments for DME, with at least 3 of these treatments being intravitreal anti-VEGF injections (excluding IAI). Treatments for DME prior to IAI switch included intravitreal bevacizumab (IVB) and ranibizumab (IVR), intravitreal triamcinolone acetonide (IVTA), sub-Tenon’s triamcinolone acetonide (STTA), dexamethasone intravitreal implant , and laser photocoagulation. If both eyes of a patient met entry criteria, then the worse-seeing eye was included in the study. If patients were treated with previous corticosteroids, baseline intraocular pressure had to be 21 mm Hg or less either with or without pressure reducing drops. Those patients who were previously treated with corticosteroids also had to have adequate clarity of media to allow adequate SD-OCT image quality. Patients were excluded if they had previously received intravitreal aflibercept in the study eye or a history of systemic anti-VEGF therapy. Inclusion and exclusion criteria are outlined in Table Intravitreal injections were carried out using the same standard procedure in all patients. Dosing of intravitreal aflibercept was administered on a monthly as needed (PRN) regimen following the initial dose of aflibercept. The PRN treatment algorithm for DME entailed treatment with intravitreal aflibercept and monthly monitoring with SD-OCT. If at any point the central macula had no fluid at all, further aflibercept injections were withheld and the patient was monitored on a monthly basis. If new or increase in fluid occurred from the previous visit secondary to worsening of diabetic macular edema, re-treatment with intravitreal aflibercept was performed.If the macula continued to have fluid after a minimum of 3 aflibercept injections and the macula achieved a 15% or more reduction in CRT compared to baseline on SD-OCT, then treatment could be withheld. If the macula continued to have fluid after a minimum of 3 aflibercept injections and the macula achieved <15% reduction in CRT compared to baseline of SD-OCT, aflibercept was continued or an alternative therapy was considered (corticosteroids or laser). If alternative therapy was utilized, the patient was not included in this analysis.Patient charts were reviewed until 6 months after the first aflibercept injection. At each visit, best-corrected distance visual acuity was measured using a standardized Snellen chart, a comprehensive eye examination was performed including intraocular pressure measurement via applanation, and OCT imaging was obtained using Spectralis OCT  or Cirrus HD-OCT .The occurrence of any severe postoperative complications, including infection, inflammation, retinal detachment, thromboembolic events, or death within the first 6 months after the first aflibercept injection was recorded.The injection logs were reviewed for all patients receiving IAI between March 1, 2014 and July 1, 2015. One hundred and forty-seven patients in total were treated with IAI during this time, and of these, 31 patients were treated with IAI for DME. Twenty patients were excluded from the study for the following reasons: 18 eyes had less than 4 treatments within the 6 months prior to switch to IAI, 6 patients had a CRT on SD-OCT of less than 350 μm at time of switch to IAI, and 2 patients had a greater than 15% decline in CRT on SD-OCT over the 6 months prior to switch to IAI. A total of 11 patients with DME meeting inclusion criteria for the study were evaluated. The mean patient age was 65 years old (range 47–83 years). There were 7 men (64%) and 4 women (36%), and 5 (45%) right eyes and 6 (55%) left eyes included in the study. The self-reported mean hemoglobin A1c was 7.2 (range 6.1–10) and the mean duration of diabetes was 15 years (range 5–34 years) at the time of enrollment in the study. Baseline characteristics are outlined in Table The average duration of treatment duration for DME prior to inclusion in the study was 32 months (range 8–77 months). The mean total number of treatments given prior to inclusion in the study was 13 (range 5–30) and included intravitreal bevacizumab, intravitreal ranibizumab, intravitreal triamcinolone acetonide, sub-tenon triamcinolone acetonide, dexamethasone intravitreal implant, and focal laser. The anti-VEGF injections given prior to inclusion in the study included ranibizumab exclusively in 4 patients (36%), bevacizumab exclusively in 2 (18%) patients, and a combination of ranibizumab and bevacizumab in 5 (46%) patients. Other treatments included intravitreal triamcinolone acetonide , sub-tenon triamcinolone acetonide , focal laser , and dexamethasone intravitreal implant .The average number of treatments for DME administered to the cohort during the 6 months before switch to IAI was 4.7 (range 4–6), with an average of 4.3 (range 3–6) being anti-VEGF injections. Eight patients (73%) were treated with ranibizumab exclusively, 2 patients (18%) were treated with bevacizumab exclusively, and 1 patient (9%) was treated with a combination of ranibizumab and bevacizumab. Other treatments included intravitreal triamcinolone acetonide  and focal laser . No patients received panretinal photocoagulation during this time.During the 6 months after switch to IAI, an average of 4.7 (range 4–6) IAI were given. All patients had 6-month follow-up after switching to IAI and this visit was used for outcome analysis. Treatment characteristics are outlined in Table The average CRT as measured on SD-OCT at 6 months prior to switch to IAI was 428 μm (range 299–690). The mean change in CRT during the 6 months prior to switch to IAI was an 18.6% increase. The average CRT at time of switch to IAI was 487 μm (range 363–685). At 6 months following switch to IAI, the average CRT was 326 μm (range 262–454). The mean change in CRT during the 6 months after switch to IAI was a 27.1% decrease. Anatomic and Snellen visual acuity outcomes before and after switch to IAI are outlined in Table At 6 months after switch to IAI, 9 of 11 patients (81%) had a 15% or more decrease in CRT, meeting the primary endpoint. At 6 months after switch to IAI, 9 of 11 patients (81%) had a 25% or more decrease in CRT and 1 of 11 patients (9%) had a 50% or more decrease in CRT. Eight of 11 patients (73%) had a CRT of less than 350 μm. Representative SD-OCT data before and after switch to intravitreal aflibercept is shown in Fig. Six months prior to switch to IAI, the Snellen visual acuity ranged from 20/25 to 20/100. Over the 6 months prior to switch to IAI, the Snellen visual acuity declined in 8 patients (73%), improved in 2 patients (18%), and remained unchanged in 1 patient (9%). At time of switch to IAI, the Snellen visual acuity ranged from 20/40 to 20/200. Six months after switch to IAI, there was some improvement in Snellen visual acuity in 7 patients (63%) and remained stable in 4 patients (36%). No patients had decline in Snellen visual acuity after switch to IAI.After inclusion in the study, 52 anti-VEGF injections were given prior to switch, and 52 aflibercept injections were given after the switch. No ocular adverse events, including endophthalmitis, uveitis, retinal tears or detachment, or elevated intraocular pressure necessitating treatment were observed. There were no systemic adverse events such as thromboembolic phenomena  during the study.Inflammatory changes in the retinal environment are involved in the pathogenesis of DME, including vascular leakage, leukostasis, ischemia, and release of pro-inflammatory mediators such as VEGF, interleukins 6 and 8, and TNF-alpha . The retIn clinical practice, some patients have chronic fluid, despite aggressive treatment with intravitreal injections of bevacizumab or ranibizumab. It is important to know if there is benefit to switching incomplete or non-responders to an alternative anti-VEGF agent, rather than continuing ranibizumab or bevacizumab injections, switching to corticosteroids, or combining anti-VEGF and corticosteroid treatment. The current study used stringent criteria identify patients with recalcitrant DME to evaluate if switching to IAI would be of benefit. In this small study, switching to IAI resulted in some anatomic improvement in all eligible patients, and 81% of patients met the pre-specified primary endpoint of 15% or greater decrease in CRT.Our results are consistent with two previously reported cases series. Wood, Karth, Moshfeghi, and Leng evaluated short-term outcomes of switching to IAI in 14 eyes with persistent fluid from DME despite at least 3 monthly ranibizumab or bevacizumab. There was no minimum central retinal thickness requirement for inclusion and follow-up occurred after a single IAI. At 1 month after injection, 79% of patients had anatomic improvement on SD-OCT .Rahimy et al. evaluated 50 eyes treated with at least 4 consecutive injections with ranibizumab or bevacizumab prior IAI switch, and two or more IAI after switch. Included patients had minimum central retinal thickness of 300 μm and follow-up occurred at month 2 or 3 after switch to IAI. At most recent follow-up, 56% had anatomic improvement on SD-OCT, while 24% had complete resolution of fluid .The greater response to aflibercept in patients with chronic DME recalcitrant to treatment with bevacizumab and ranibizumab may be due to a number of factors, and may help explain the results seen in Protocol T. Aflibercept has been shown to have significantly higher binding affinity to VEGF-A compared to ranibizumab and bevacizumab. Also, aflibercept binds to VEGF-B and placental growth factor (PlGF), unlike ranibizumab and bevacizumab . These cIt is unclear both from inconsistencies in the published literature and clinically as to what parameters should be used to define recalcitrant DME . The current study used pre-defined and very stringent criteria for classifying patients as having recalcitrant DME, and included visual acuity, central retinal thickness, and treatment parameters. Recalcitrant patients were identified as those with substantially thickened retinas (>/350 μm CRT) with minimal improvement in CRT (<15% across 6 months prior to switch) despite aggressive therapy with at least four treatments for DME (3 of which were VEGFs other than aflibercept). These eyes were not improving anatomically prior to switch despite frequent anti-VEGF therapy. Switching to a pre-defined minimal use of aflibercept resulted in some anatomic improvement in all eligible patients. All patients were followed for at least 6 months, with the majority of patients gained some visual acuity after switching to aflibercept, suggesting some reversibility of functional damage.The major limitations of this study include the retrospective design and the relatively small number of participants, precluding the ability to apply statistical analyses. Further studies are needed on a larger scale but this small, retrospective study suggests that switching to aflibercept can be beneficial for recalcitrant DME patients as defined in this study.In conclusion, in patients with recalcitrant DME, defined as having thickened retinas (≥350 μm) with minimal decrease in CRT (<15% over 6 months prior to switch) despite aggressive treatment , switching to intravitreal aflibercept may result in improved anatomic and visual outcomes at 6-month follow-up.


Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen.Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity.Results: Three of the four lines  were triple negative , with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 (CA9) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B, and PMS2, which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O2) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen. This is considerably higher than that %. This i tissues , 4. In od damage , 7 and m range 1–% as a co2, 5% CO2, 90% N2) to mimic physiologically oxygen levels in tumors. The cell lines of ATCC provenance were cultured in an atmosphere of 5% CO2 in air at 37°C.Culture conditions have been described , 10 for For estrogen response, MCF-7 and NZBR3 cell lines were cultured in estrogen deprived media , and penicillin/streptomycin  for the 2 days before 17β-estradiol treatment. Estrogen-deprived medium was used for all assays related to estrogen stimulation.As described previously , tumor t2, 5% CO2, and 90% N2 in a Tri-Gas Forma incubator. Tissue culture plates or flasks had previously been coated with a thin layer of agarose to prevent the growth of fibroblasts . Material containing larger aggregates was pipetted into tubes, collected by low-speed centrifugation to remove blood cells, necrotic material, and debris , and then washed twice  in growth medium. Preparations were monitored by phase contrast microscopy, and cytospins of cell suspensions were stained by hematoxylin/eosin and examined by a pathologist to ensure that they contained tumor cells. Cultures were set up in growth medium supplemented with 5% FBS under an atmosphere of 5% OEarly passage cell lines (< passage 33) used in this study were developed in this laboratory. All cell lines were tested negative for mycoplasma contamination.Everolimus, afatinib, dacomitinib, lapatinib and ARRY-380 was purchased from Selleck Chemicals . 17β-estradiol, tamoxifen, paclitaxel and doxorubicin were purchased from Sigma . NVP-BEZ235 , 12 was The NZBR cell lines were typed by short tandem repeat profiling by DNA Diagnostics  Table . The com3H-thymidine (0.04 μCi per well) was added to each well and cultures were incubated for 6 h; cells were harvested on glass fiber filters using an automated TomTec harvester. Filters were incubated with Betaplate Scint and thymidine incorporation measured in a Trilux/Betaplate counter. Effects of inhibitors on the incorporation of 3H-thymidine into DNA were determined relative to the control (non-drug-treated) cells.As described previously , 13, 14,For the growth response to 17β-estradiol in 5 and 21% oxygen culture conditions, cells were seeded at 2,500 per well in 96 well plates in the presence or absence of 50 nM 17β-estradiol for 3 days. For the growth response to doxorubicin in 5 and 21% oxygen culture conditions, cells were seeded at 1,000 per well in 96 well plates in the presence of varying concentrations of inhibitors for 5 days.5) were harvested, incubated with 20 μM DCFDA in medium for 60 min in the dark at 37°C. Cells were analyzed in a BD Accuri™ Flow Cytometer.Intracellular ROS were detected with the cell-permeable fluorescent probe 2',7'- dichlorofluorescein diacetate (DCFDA) (ABCAM) according to the manufacturer's instruction. Cells (1 × 10CA9), which is the most well established target of HIF, quantitative RT-PCR (qRT-PCR) was performed using gene-specific primers (Table HPRT1 RNA expression. The 2∧(–delta delta CT) method was used to analyze the relative changes in gene expression.As described in detail , oligo-drs Table  and SybrAs described , 10, 16,For whole exome sequencing (WES), 250 ng of genomic DNA from each cell line was sheared using the EpiShear™ Multi-Sample Sonicator (Active Motif). The quantity and fragment size of the sheared DNA were assessed on a Tapestation 2200 (Agilent) with the high sensitivity D1000 tape. Sheared DNA (100 ng) was used for the preparation of the whole exome libraries (WELs). WELs were prepared using the SureSelect XT2 (SSXT2) reagent kit and the SureSelect Clinical Research Exome V2 exome enrichment kit following the manufacturer's instructions (Agilent Technologies). The WELs were sequenced on a NextSeq500  to obtain around 40 to 44 million paired end reads  per exome.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were aligned to the human reference genome (hg19) with BWA (bwa 0.7.12)  . The res 0.7.12) . Mpileupgenome hg with BWA 0.7.12) . The var 0.7.12)  followed 0.7.12) . Variant 0.7.12) .T-tests or Mann-Whitney Rank Sum Tests was used for comparison of groups. Correlation analysis was performed with Spearman Rank Order correlation coefficient (R) and statistical significance (P) using SigmaPlot . Values of P < 0.05 were considered to be statistically significant.The cell lines were characterized by cellular DNA content, hormone receptor expression and tamoxifen sensitivity. The lines were all aneuploid and three of the four lines  were triple-negative with no expression of estrogen receptor, progesterone receptor and HER2 Table . The ER+3H]-thymidine incorporation assay was used to assess the effect of estrogen on cell proliferation when the cells were cultured under 5 and 21% oxygen conditions. Both MCF-7 and NZBR3 cell lines displayed significant growth stimulation by estrogen and MCF-7 cells showed increased response to estrogen as compared to NZBR3. However, the two oxygen concentrations did not have distinguishable effects on the growth response to estrogen , Cyclin D1 (CCND1) and Trefoil Factor 1 (TTF1)  . Both GRn Figure .T- test, p < 0.05)  in cells cultured under 5 and 21% oxygen concentrations. A significant increase in ROS concentrations was observed in cells cultured in 21% oxygen (pairwise ) Figure .CA9) -regulated genes, including carbonic anhydrase IX (CA9) . Cell lin Figure .The phosphorylation status of AKT, p70S6K, rpS6, and ERK in the NZBR lines was examined Figure . NZBR1 sWe have shown that estrogen receptor positive (ER+) breast cancer cell lines generally are more sensitive to everolimus than are receptor negative lines . However3H-thymidine incorporation assay. The ER+ HER2+ NZBR3 cells showed high sensitivity to everolimus (IC50 11.5 nM), yet the triple-negative NZBR2 with low mTOR pathway utilization also showed increased everolimus sensitivity (IC50 1.1 nM)  between NZBR2 and the other three cell lines in BEZ235 sensitivity were also observed.Two out of the three triple-negative NZBR cell lines were resistant to everolimus, with IC50 values of over 500 nM Figure  in a 3-d) Figure . SignifiThe sensitivities of the NZBR cell lines, and the ER+ MCF-7, HER2+ SKBR3 and triple-negative MDA-MB-231 cell lines to the therapeutic agents doxorubicin (a topoisomerase II poison) and paclitaxel (a microtubule poison) were tested Figure . The ER+T-test, p > 0.05) in sensitivity to doxorubicin was observed when the cell lines were cultured in 5% vs. 21% oxygen . As expected, drug resistance was observed in the HER2- cell lines; however, the HER2+ cell line ZR75.1 also showed similar resistance Figure . HER2+ NThe effects of ARRY-380 on HER2, AKT, and ERK activation were examined in four HER2+ breast cancer cell lines, which showed a range of sensitivities to ARRY-380. After overnight exposure to 100 nM or 1000 nM of ARRY-380, we observed no effect on total HER2, AKT and ERK expression in the cell lines. ARRY-380 inhibited HER2 phosphorylation in all lines, and ERK phosphorylation in cell lines with detectable phosphorylation Figure . Of inteAKT1, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, FANCC, MEN1, MRE11A, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, ALB2, PIK3CA, PMS1, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCCC2) were selected from the hereditary breast cancer panel of AmbryGenetics, GeneDx, and University of Washington. The somatic mutations gene list  was selected from somatic mutations in the cancer (COSMIC) database. Several sequence variants that result in missense or nonsense mutations were detected in genes associated with breast cancer  gene lies within an intron of the NF1 (Neurofibromatosis type 1) gene and is transcribed in the opposite direction to the NF1 gene (NF1 and EVI2B RNA expression levels in relation to those of the estrogen receptor gene ESR1 in the genome-wide RNA transcript profile from TCGA (breast invasive carcinoma gene expression) using the RNAseq data set . A weak but significant positive correlation was observed in the expression of ESR1 and NF1  while EVI2B and ESR1 expression were negatively correlated . No significant correlation of NF1 and EVI2B was observed .Most mutated genes found in the NZBR cell lines had also been found to be mutated in other breast cancer cell lines Table . The muts. Table . Since tNF1 gene , we comp2) is generally similar to that of commercially available breast cancer cell lines. It is of interest that the hormone receptor status of the new lines covers the main classes of breast cancer, with three of the four having triple negative receptor status. Proliferation of the ER+ NZBR3 cell line was sensitive to estrogen stimulation, and expression of the estrogen responsive genes GREB1 and TFF1 was increased after 24 h of estrogen exposure   of a cell line cohort (TP53 mutations is lower in cell lines derived under low oxygen conditions.The genomic mutation frequency in the breast cancer cell lines is significantly higher than that in breast carcinoma 2) Table , but thees Table . The avae cohort . HoweverThe triple negative line NZBR1 is of interest because of the relative absence of gene mutations in the genome sequence. One possible explanation is that this cell line possesses a CpG island methylator phenotype (CIMP) –16 that BRCA2 mutation as well as an AKT1 mutation. The latter differs from the hot spot mutation AKT1E12K that confers sensitivity to the dual mTOR/PI3K inhibitor BEZ235 , mismatch repair system component homolog 2 (PMS2), MutL homolog 1 (MLH1) and Ataxia Telangiectasia Mutated serine/threonine kinase (ATM). BRCA2, a tumor suppressor that is essential for the repair of double-strand DNA breaks by homologous recombination, is associated with risk of developing breast cancer , EGFR (L858R), EGFR (L858R/T790M) and HER2 with IC50s of 0.5, 0.4, 10, and 14 nM respectively in cell-free assays. Dacomitinib is a potent, irreversible pan-ErbB inhibitor, mostly of EGFR with an IC50 value of 6 nM in a cell-free assay. ARRY-380 is a potent and selective HER2 inhibitor with IC50 of 8 nM, equipotent against truncated p95-HER2, 500-fold more selective for HER2 over EGFR . ARRY-380 is under clinical investigation for treating HER2+ metastatic breast cancer patients. Since ARRY-380 is HER2 specific, it has the potential to block HER2 signaling without causing the toxicities of EGFR inhibition . In comp2) is generally similar to that of commonly used breast cancer cell lines developed under 21% oxygen conditions. Although no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5% vs. 21% oxygen, reduced ROS and upregulation of the hypoxia response, as indicated by CA9 expression, were observed in cells cultured under 5% oxygen conditions. Therefore we cannot exclude the possibility that culture of cells under 5 vs. 21% oxygen conditions could have consequences for the effectiveness of drug treatment in cell lines as predictors of therapy responses in patients.In summary, our results suggest that the behavior of cell lines developed under low oxygen conditions (5% OEthics approval and consent to participate: This study was carried out in accordance with the recommendations of New Zealand Health and Disability Ethics Committee guidelines–ref AKL/2000/184/AM06, New Zealand Health and Disability Ethics Committee. The protocol was approved by the Northern A Health and Disability Ethics Committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki.EYL and BCB designed this study. EYL, CF-P, WRJ, EM, PMK, DCS and MEA-A carried out the study. EYL, CF-P, SKB, RJB, DCS and MEA-A assisted with the data analysis. EYL drafted the manuscript. RJB, GJF, DCS, CF-P, MEA-A, SKB and BCB assisted with the manuscript preparation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although hatching is perhaps the most abrupt and profound metabolic challenge that a chicken must undergo; there have been no attempts to functionally map the metabolic pathways induced in liver during the embryo-to-hatchling transition. Furthermore, we know very little about the metabolic and regulatory factors that regulate lipid metabolism in late embryos or newly-hatched chicks. In the present study, we examined hepatic transcriptomes of 12 embryos and 12 hatchling chicks during the peri-hatch period—or the metabolic switch from chorioallantoic to pulmonary respiration.THRSPA. Using pairwise comparisons of embryo and hatchling ages, we found 1272 genes that were differentially expressed between embryos and hatchling chicks, including 24 transcription factors and 284 genes that regulate lipid metabolism. The three most differentially-expressed transcripts found in liver of embryos were MOGAT1, DIO3 and PDK4, whereas THRSPA, FASN and DIO2 were highest in hatchlings. An unusual finding was the “ectopic” and extremely high differentially expression of seven feather keratin transcripts in liver of 16 day embryos, which coincides with engorgement of liver with yolk lipids. Gene interaction networks show several transcription factors, transcriptional co-activators/co-inhibitors and their downstream genes that exert a ‘ying-yang’ action on lipid metabolism during the embryo-to-hatching transition. These upstream regulators include ligand-activated transcription factors, sirtuins and Kruppel-like factors.Initial hierarchical clustering revealed two distinct, albeit opposing, patterns of hepatic gene expression. Cluster A genes are largely lipolytic and highly expressed in embryos. While, Cluster B genes are lipogenic/thermogenic and mainly controlled by the lipogenic transcription factor ectothermy (embryo) to endothermy (hatchling) in the chicken. Several transcription factors and their coactivators/co-inhibitors appear to exert opposing actions on lipid metabolism, leading to the predominance of lipolysis in embryos and lipogenesis in hatchlings. Our analysis of hepatic transcriptomes has enabled discovery of opposing, interconnected and interdependent transcriptional regulators that provide precise ying-yang or homeorhetic regulation of lipid metabolism during the critical embryo-to-hatchling transition.Our genome-wide transcriptional analysis has greatly expanded the hepatic repertoire of regulatory and metabolic genes involved in the embryo-to-hatchling transition. New knowledge was gained on interactive transcriptional networks and metabolic pathways that enable the abrupt switch from The online version of this article (10.1186/s12864-018-5080-4) contains supplementary material, which is available to authorized users. The developing mammalian embryo is completely dependent upon its mother and her placenta to supply nutrients, exchange respiratory gases, and to remove nitrogenous waste products. Quite the opposite is true among amniotes , which must undergo independent embryonic development in a pre-formed and completely enclosed system—the cleidoic egg. In chickens, the principal source of energy for late embryonic growth is derived from yolk lipids stored in the egg by the hen. The embryo exhibits exponential growth between days 13 to 18 of embryonic development (E13 to E18), when the total energy required for growth comes from β-oxidation of fatty acids derived from yolk lipids . The finDIO3) at E20, whereas the activity of the activating deiodinases (DIO1 and DIO2) increases in E20 embryos during hatching and afterwards in the hatchling. The pro-thyroid hormone (T4) is readily deactivated to reverse rT3 by DIO3 in liver of E16 and E18 embryos; then with onset of pulmonary respiration, the activity of (DIO1) and (DIO2) increases in liver to meet the ensuing energy demands of hatching and endothermy. The embryo must switch from chorio-allantoic to pulmonary respiration and instantly meet the high metabolic demands of an endotherm—the hatchling chick. Therefore, it is essential to understand the transcriptional networks that integrate and control function of the endocrine system to meet the dynamic metabolic demands of growth imposed upon the liver.The chicken embryo develops as an aquatic ectotherm with respiratory gas exchange via the chorioallantoic membrane. On the last day of embryonation (E21), the fully-developed embryo must suddenly convert to pulmonary respiration after pipping through the eggshell. During the peri-hatch period, the precocious chick becomes an endotherm with a very high and self-sustaining metabolic rate maintained by a fully-functional thermoregulatory system. The thyroid axis plays a major role in regulating metabolism and energy expenditure. Hence, the activity and importance of the thyroid axis has been extensively studied during the peri-hatch period of the chicken –18. The see Fig. THRSPΑ, SCD, FASN, ME1, HMGCS, and EVOL6).Our first glimpse of the liver transcriptome during the chicken’s peri-hatch period came from an earlier low-resolution transcriptional scan using the original chicken liver 3.2 K) microarray . This in K microaectothermy (embryo) to endothermy (hatchling) in the chicken.The present high-resolution genome-wide transcriptional analysis of the liver during the peri-hatch period has greatly expanded our hepatic repertoire of regulatory and metabolic genes involved in the embryo-to-hatchling transition. Several transcription factors were identified that appear to exert opposing actions, which lead to the predominance of lipolysis in embryos or lipogenesis in hatchlings. Furthermore, we have gained new knowledge on interactive transcriptional networks and metabolic pathways that enable the abrupt switch from Fertile eggs from Ross x Ross broiler (meat-type) chickens were purchased from a commercial hatchery . The eggs were incubated in an automated incubator  at 39 °C and 95% relative humidity. At E20, eggs containing viable embryos were transferred to hatching trays to determine the approximate time of hatch . Chicks hatched within a 4 h period on the 21st day of incubation were wing-banded and held in the incubator for an additional 4 h. The remaining one day-old (D1) chicks were vaccinated against Marek’s disease virus and transferred to a heated battery brooder  maintained at 33 °C. The chicks received a commercial starter ration  and water ad libitum throughout the experiment. After continuous light for the first two days, the birds were maintained on a 20 h light: 4 h dark (20 L:4D) photoperiod. All protocols used in this study were in accordance with the United States Department of Agriculture guidelines on the use of agricultural animals in research and approved by the University of Delaware Animal Care and Use Committee.The experimental design used for the present transcriptional profiling with Affymetrix Chicken Genome arrays was similar to the design utilized in our original 3.2 K microarray analysis of liver in peri-hatch chickens . Four maTotal RNA was purified from each liver sample using an RNeasy Mini kit with on column DNase I treatment, as recommended by the manufacturer’s . Quantitation of total RNA was done using a NanoDrop spectrophotometer  and the RNA was diluted to a standard concentration (250 ng/μL). RNA quality was assessed using an Agilent 2100 Bioanalyzer and an Agilent RNA 6000 Nano Kit . Liver RNA samples (1 μL each) were analyzed on a RNA 6000 Nano Chip. The ribosomal RNA ratio (28S/18S) was assessed for RNA integrity, where with a RNA Integrity Number (RIN) of 9 to 10 indicates RNA of high quality for use in amplification of RNA for the microarray analysis and for verification by qRT-PCR analysis. Each liver RNA sample was divided into two identical aliquots and stored frozen at − 85 °C until use in the transcriptional analyses below.Microarray-based transcriptome profiling was carried out using 24 Affymetrix GeneChip™ Chicken Genome Arrays and one-cycle target labeling protocol according to the manufacturer’s (Affymetrix) protocols. The experimental design for hybridization of the 24 chicken genome arrays was based on four replicate hybridization days, where all six ages were represented by one of four biological replicates  per age group  of the microarray data was completed using GeneSpring GX software . Raw cell files from the Affymetrix GeneChip Chicken Genome Arrays were imported into GeneSpring software and unfiltered differentially-expressed genes  identified for a preliminary hierarchical and k-means cluster analysis  and a log2 ratio ± 0.75 cutoff. This gene expression level threshold of log2 ratio ± 0.75 (or a 1.68-fold difference) has proven useful for statistical analysis of microarray data in model plants  control expression of multiple metabolic enzymes , kinases , a phosphatase (LPIN1), and transporters (STARD4 and SLC6A6). The gene network in Fig. PPARG, PPARGC1A, NR0B1, NCOR1and ZBTB20) with key lipogenic metabolic enzymes , growth factors (IGF1), binding proteins  and the adiponectin receptor (ADIPOR2). Other direct targets of PPARG shown in this gene network were NDRG1, PLIN2, PER3 and VNN1.Two gene interaction networks from the D1 vs. D9 contrast were functionally annotated by IPA as involved in “Lipid Metabolism” Fig. . As showTHRSPA, PPARG, PPARD and KLF15), where PPARG and PPARD share seven target genes . The TF PPARG and several of its direct targets are expressed at higher levels in liver of D9 hatchlings, including ADIPOR2, FBXO9, ELOVL6, ELOVL2, ELOVL1, HSD17B12, PEX11A, HECTD2, VNN1 and FDPS. The fasting-induced transcriptional factor KLF15 and four direct gene targets  are expressed higher in liver of fasted D1 hatchlings. The Ingenuity Upstream Regulator Analysis , since 21 of its target genes are down-regulated (green symbols) AR-DEGs in this contrast. Based on known relationships in the Ingenuity Knowledge Base, Ingenuity predicts that inhibited PPARG would block the expected activation (blunt orange lines) of CDK1, CPT1A and LPL. Ingenuity predicts that KLF15 would be activated, which would lead to activation (up-regulation) of seven direct targets  as indicated by the orange arrows. Interestingly, KLF15, a key transcriptional regulator of gluconeogenesis, was highly expressed in liver of fasted D1 hatchling chicks, as revealed by three pairwise contrasts .The final gene interaction network Fig.  was alsosis Fig.  identifiCOX7A2L) during the embryo-to-hatchling transition were examined by qRT-PCR analysis  show higher log-based expression in embryos and a sharp decline after hatching. Surprisingly, hepatic expression of DIO1 was relatively stable, except in E18 embryos where DIO1 was slightly elevated. The invariant gene (COX7A2L) was used by geNorm software for normalization of the qRT-PCR expression levels. The expression patterns of eight lipogenic genes during the embryo-to-hatching transition are depicted in Fig. THRSPA and SCD was log-scale and increased exponentially, reaching a plateau in liver of fed hatchlings between D3 and D9. Similar log-scale expression patterns were found for fiv other metabolic genes , while HMGCL and HMGCS2 only showed a slight increase in transcript abundance in D9 hatchlings.Hepatic expression patterns of seven DEGs that were expressed higher in embryos and an invariant gene ]. The deactivating deiodinase DIO2 and the adipokine ADIPOQ showed a progressive increase in hepatic expression from E16 to a peak in E20 embryos. Although DIO2 levels were lower in D1 and D3 hatchlings, a peak in abundance was reached in D9 hatchlings. The qRT-PCR analysis . Five hepatic genes  had higher expression levels, albeit with a lower amplitude, in D3 and D9 hatchlings. We were surprised to find that hepatic expression of DIO1 was rather stable across embryonic (E16-E20) and hatchling (D1-D9) ages. Furthermore, DIO1 was not detected as a DEG by genome array analysis or qRT-PCR analysis; therefore, DIO1 was considered an invariant gene and not included in the Pearson’s Correlation Analysis.Figure sis Fig. , bottom sis Fig. , top disr = 0.929; 11 degrees of freedom) indicates a highly significant (P ≤ 0.01) correlation between gene expression levels obtained from both microarray and qRT-PCR analyses  which interact with each other and their respective target genes in late embryos as they utilize yolk lipids and prepare for hatching on E21. Upon feeding, hatchling chicks abruptly increase expression of several ligand-activated transcription factors  which in turn control numerous down-stream metabolic enzymes, transporters and kinases/phosphatases to support greater rates of energy expenditure for lipogenesis, adipogenesis and somatic growth. Hierarchical clustering revealed two opposing patterns of gene expression in liver during the peri-hatch period, where Cluster A had higher expression in embryos, which sharply declined after hatching  provides a detailed view of the innate choreography of major transcriptional responses involved in the abrupt switch from a lipid-laden ectotherm (embryo) to a free-living endotherm (hatchling chick). We have identified several transcription factors  for rapid growth and metabolism in hatchling chicks  were up-regulated in liver of D9 hatchlings with direct interactions with SREBF2 and ACACA, which encodes the rate limiting enzyme in fatty acid synthesis. Apparently, both THRSP and MID1IP1 are glucose-responsive genes, which have overlapping roles in regulating expression of several lipogenic genes  had 19-times greater abundance in liver of riboflavin-rescued embryos than riboflavin-deficient embryos between E13 and E15. Thus, three independent chicken microarray platforms and multiple expressed-sequence tag (EST) sequences in GenBank clearly support abundant “ectopic” expression of FKER in liver of E15 and E16 embryos from both egg-type (Leghorn) [FKER in the lipid-laden liver of late embryos and the relationship between hepatic expression and cutaneous expression of FKER in embryos will require further definitive study.Interestingly, d GGA27) –67. Fiven rescue . We propatocytes . Furtherabolites . Examinarop milk . The unir THRSPA . The pre embryos , which cGPL1742) . This cDLeghorn)  and, prePPARA, PPARGC1A, NR1H4 and SIRT1, SERTAD2, KLF11, KLF13 and KLF15) and scores of metabolic enzymes, transporters, phosphatases and kinases. The metabolites derived from these processes interact with nuclear receptors, G-coupled protein receptors (GCPRs), transmembrane receptors and extracellular factors. Highly-expressed hepatic DIO3 inactivates thyroid hormones in the embryo, while its expression sharply declines in hatchling chicks. Although hepatic expression of DIO1 was rather constant during the peri-hatch period, another thyroid hormone-activating enzyme DIO2 reached peak abundance at two ages, E20 and D9. Upon pipping through the egg shell, the hatching chick immediately converts to the pro-hormone T4 into metabolically-active T3, which supports achievement of thermodynamic freedom in hatchlings and synergetic activation of THRSPA, the major lipogenic transcription factor in chickens. Furthermore, T3 bound to its receptor (THRB) forms heterodimers with other nuclear receptors, which then control programmed expression of major lipogenic enzymes . In addition to THRSPA, other transcriptional regulators  also interact and contribute to the sharp increase in lipogenesis observed in liver of D3 and D9 hatchlings.The present analysis of the embryo-to-hatchling transition in meat-type chickens provides the first detailed view of the choreography of innate and dynamic transcriptional responses made by embryos and newly hatched chicks during the critical metabolic jump from lipid-laden ectotherm to free-living self-sufficient endotherm, respectively. The metabolism of late (E16-E20) embryos is dominated by β-oxidation of yolk lipids, glycolysis and gluconeogenesis, which utilizes the residual albumen and other proteins in allantoic fluid ingested by the embryo just prior to hatching. These metabolic processes are precisely controlled by multiple and interacting transcription factors  chickens. We have proposed that hepatic expression of FKER in e15-e16 embryos could be an adaptive response to the coincident and massive accumulation of yolk lipids. This idea is supported by the exceptional expression of multiple β-keratin transcripts, including FKER, in the lactating crop sac of the pigeon [THRSPA. Nevertheless, the biological function of FKER expressed in liver of e15-e16 embryos and the relationship between hepatic FKER and cutaneous FKER remain to be elucidated by definitive study. The present study provides new insight into dynamic interaction of multiple transcription factors and their direct target genes that provide homeorhetric regulation of metabolism during the abrupt embryo-to-hatchling transition of the domestic chicken, Gallus gallus.Our present observation of the extraordinary expression of multiple e pigeon , which aAdditional file 1:Experimental design of microarray hybridizations. A Microsoft Excel file containing a single work sheet “Array Hybridization Design” which describes the hybridization scheme used for the 24 Affymetrix Chicken Genome Arrays. (XLSX 12 kb)Additional file 2:Primers used for qRT-PCR analysis. A Microsoft Excel file containing a single work sheet “qRT-PCR Primer Information”. This table provides the chicken gene symbol, GenBank ID number, 5′-3′ sequence for forward and reverse primers, and amplicon size (bp) for each gene used for qRT-PCR analysis. (XLSX 12 kb)Additional file 3:P ≤ 0.05; ±0.75 cutoff) for each contrast. (XLSX 905 kb)Annotated gene list for 14 pairwise contrasts of liver transcriptomes in embryos and hatchlings across six ages during the peri-hatch period (E16-D9). The last worksheet provides the list of 1005 commonly-shared AR-DEGs identified by the Venn diagram shown in Fig. Additional file 4:P ≤ 0.05) that AR-DEGs belong to a particular canonical pathway or biological function accrued in the Ingenuity Knowledge Base. (XLSX 53 kb)Canonical and biological pathways identified by the contrast of all embryos vs. all hatchlings. The AR-DEGs from the E vs. H contrast were assigned by Ingenuity Pathway Analysis (IPA) to canonical and functional and pathways. Fisher’s Exact Test is used in IPA to determine probability  of each AR-DEG belonging to a particular canonical pathway or biological function. (XLSX 36 kb)Canonical and biological pathways identified by IPA in the E18 vs. E20 contrast. The AR-DEGs from the E18 vs. E20 contrast were assigned by IPA to overrepresented canonical and functional pathways. IPA uses Fisher’s Exact Test to determine probability  of each AR-DEG belonging to a particular canonical pathway or biological function. (XLSX 44 kb)Canonical-biological pathways identified by IPA in the contrast of E18 embryos vs. D3 hatchlings. The AR-DEGs identified in the E18 vs. D3 contrast were assigned by IPA to overrepresented canonical and functional and pathways. IPA uses Fisher’s Exact Test to determine probability  of each AR-DEG belonging to a particular canonical pathway or biological function. Each worksheet provides a list of AR-DEGs assigned to particular canonical pathways or biological functions, annotated with gene symbol, Entrez name, log2 ratio, cellular compartment, type and Entrez protein ID. (XLSX 31 kb)Canonical and biological pathways found by IPA in the D1 vs. D9 hatchling contrast. The AR-DEGs identified in the D1 vs. D9 contrast were assigned by IPA to overrepresented canonical and functional and pathways. IPA uses Fisher’s Exact Test to determine probability  indicates a highly significant correlation between gene expression levels obtained from both microarray and qRT-PCR analyses. (XLSX 16 kb)Pearson’s correlation analysis of gene expression levels of 15 “candidate” DEGs determined by genome microarray and verified by qRT-PCR analyses. A Microsoft Excel file containing the average normalized expression levels (log2 ratios) of 15 candidate DEGs as determined by genome microarray analysis and verified by qRT-PCR analysis. The Pearson’s correlation coefficient (


Slow tool servo (STS) diamond turning is a well-developed technique for freeform optics machining. Due to low machining efficiency, fluctuations in side-feeding motion and redundant control points for large aperture optics, this paper reports a novel adaptive tool path generation (ATPG) for STS diamond turning. In ATPG, the sampling intervals both in feeding and cutting direction are independently controlled according to interpolation error and cutting residual tolerance. A smooth curve is approximated to the side-feeding motion for reducing the fluctuations in feeding direction. Comparison of surface generation of typical freeform surfaces with ATPG and commercial software DiffSys is conducted both theoretically and experimentally. The result demonstrates that the ATPG can effectively reduce the volume of control points, decrease the vibration of side-feeding motion and improve machining efficiency while surface quality is well maintained for large aperture freeform optics. Most currently conventional optical devices from 2D optical design methods are usually rotationally symmetric. While systems with such optics perform well in numerous applications, the requirement of high energy efficiency or aberration correction often cannot be satisfied with the same optics because of its inherent symmetry limitations on geometry. In order to meet those kinds of demand, 3D optical design methods have been developed, such as the simultaneous multiple surface (SMS) 3D method , supportOver the past decades, much research work has been conducted on tool path generation for diamond turning of freeform optics, including angle sampling strategy, spiral path and tool radius compensation. Fang et al.  used nonx-axis) when it follows the c-axis. Due to the limitation of the dynamic response of the servo axis, this will lead to the following error and vibration.During the conventional multi-axis computerized numerical control (CNC) milling of freeform surfaces, it is crucial to adapt the side-feeding motion and forward sampling of tool motions to surface shape variations to achieve uniform surface quality with maximum efficiency . Koren eGenerally, the following problems exist in tool path generation algorithm for slow tool servo diamond turning of large aperture freeform optics:Current angle sampling strategies are still constant-angle and constant arc-length methods ,16, whicThe cutting residual is caused by the tool radius envelope along the feeding direction, which is regarded as surface roughness. Kwok et al.  and Yu eCurrent tool path generation methods do not consider the dynamic performance of machine tools. In traditional machining of rotational symmetrical surfaces, the feed axis and spindle are independent, so the dynamic performance of machine tool is not considered. However, when a freeform surface is machined by STS diamond turning, the feed axis and the spindle should maintain a strict spatial position relationship. Therefore, an optimal tool path is very important. Although the ATS method proposed by Zhu  can prodIn this paper, a novel path generation of STS diamond turning for large aperture freeform optics; namely, adaptive tool path generation (ATPG) is proposed. Firstly, the principle of the novel generation method is introduced and the path generation algorithm is presented in detail with analysis of the motion characteristics of machine tools. The results show that, on the premise of satisfying the machining accuracy, the proposed algorithm can reduce the volume of control points, and improve the dynamic characteristics of machine tools and machining efficiency. Finally, both theoretical analysis and experimental comparison of the algorithm are conducted.z-axes of machine tools (STS).The principle of diamond tool turning freeform optics is that the diamond tool contours the complex surface along the spiral path as shown in Lθ between the two consecutive control points as well as the local surface profile. Generally, most diamond machine tools offer PVT spline interpolation for STS, which creates a path known as a cubic Hermite Spline. Therefore, for a given interpolation tolerance εf, the algorithm adaptively adjusts the angle interval Lθ according to the error of Hermite interpolation to ensure that the error is within the range , so that the error is homogeneously distributed in the whole cutting direction.There are two intrinsic errors in STS machining, namely interpolation error and cutting residual as shown in 1 and θ2 of the same radius is different, so the calculated feedrate f1 and f2 on the same radius are different, which leads to the position of diamond tool changing with spindle angle in the process of feeding. As a result, micro-fluctuations are generated in the process of machining, which would deteriorate the surface quality of machining, just as shown in In the feeding direction, in order to improve the machining efficiency, the feedrate should be adjusted with surface curvature. Because the curvature of a freeform surface not only changes with radius, but also changes with polar angles, as shown in Although the feedrate in ATPG is slightly smaller than ATS, it is still larger than the traditional settings. The advantages of this setting are that it not only improves the machining efficiency, but also suppresses the micro-fluctuation caused by surface shape. Although this will result in a slight heterogeneous distribution of interpolation error at different angles on the same radius, it is almost negligible, and the advantage is to increase the stability of the machine tool.In the traditional STS diamond turning, the machined surfaces have to be evaluated after the machining process which gives a high risk of the machined surface failing to meet the contour accuracy. The surface generation algorithm is not only the generation of a diamond tool path, but also a predictive method to analyze the contour accuracy by using different cutting strategies.Due to the geometrical complexity of freeform surfaces, it is necessary to check tool interference, and the sectional curve method  is used z = f, which can be also expressed as z = g by coordinate transformation.The coordinate system of the workpiece for STS machining is shown in iN-th revolution of the spiral path is ρi at the angle θi = 2πi, so the corresponding curvature at this revolution can be defined as , and ϕ(4)(∙) denotes the 4th derivative.PVT mode provides an excellent contouring capability because it takes the interpolated commanded path exactly through the control points. It generates a path known as a Hermite spline. Hence, according to the error of piecewise cubic Hermite interpolation, the relationship between the interpolation error tolerance εucted by :(10)εf≤LLθ and θk+1 be derived from Equations (10) and (11) by numerical calculation.The following relation is also established as:kN+1 for kP+1 can be obtained by:The corresponding revolution kP+1 can be obtained by:The other parameters ρ for kP+1 can be obtained by combining the equations from Equation (8) to Equation (14). By conducting the iterative steps with respect to N1 = 0, the whole toolpath can be adaptively resampled.Thus, the coordinate of point xz-direction compensation , and the interpolation errors are homogeneously distributed over the entire surface. For DiffSys which adopts the constant-angle methods, the surface quality of central region is better than that of the outer region, and in order to ensure the allowable interpolation error on the outer region, a large number of sampling points are needed on the outer region of the surface, which results in redundant control points on the center region. In this case, the number of control points in ATPG  was only about 64% of that in DiffSys (6061). Therefore, the machining time in DiffSys would be much longer than that in ATPG to have similar machining accuracy.We computed the intermediate “way-points” for each axis for each point along the spline path by Hermite spline interpolation. After subtracting the desired surface, error maps of the theoretically generated surface for ATPG and DiffSys are illustrated in f = 8.9 μm/rev, which was based on the surface finish specification and the maximum slope of the part, and the tool radius compensation in DiffSys was set along the z-direction as well.The astigmatic (AST) surface was machined for investigating the performances. Considering the testing ability of the interferometer, the aperture of the workpiece was 50 mm, the amplitude was set as 4.8 μm, respectively. To have a comparison, the tool path was generated by both ATPG and DiffSys, and DiffSys follows the constant-angle strategy that was based on the interpolation tolerance and the minimum radius of curvature of the part. The feedrate in DiffSys was set as In order to validate the performance of ATPG for large aperture optical surfaces, an off-axis paraboloid (OAP) with an aperture of 213 mm was also machined, where the vertex curvature radius R was 915 mm and off-axis magnitude was 915 mm. We compared the programs generated both by ATPG and Diffsys for the same setting rule.® 250 ultra, Keene, NH, USA). The configurations of this experiment were shown in The cutting experiments were performed on a CNC ultra-precision lathe  was employed to measure the machined surface form error. The AST surface was tested directly by the interferometer and the OAP was tested though optical properties of quadric surfaces as shown in After subtracting the design surface, the form errors for the two machined surfaces (AST) are respectively shown in The machined OAP surfaces were measured through optical properties of quadric surfaces as shown in Generally speaking, more control points mean higher interpolation accuracy. However, as the number of points increases, the number of points is no longer the bottleneck for machining accuracy. Furthermore, because the new tool path makes the axis speed variation very small, the machine tool can adapt to a higher spindle speed. Hence, the comparison verifies the high machining efficiency of the ATPG for achieving the same surface quality. It is noted that the PV value of the form error and roughness is higher than the preset theoretical one. The reason is that, besides the accuracy of interpolation, factors that determine the surface form and roughness still include machine tool vibration, tool wear, lubrication and clamping deformation, etc.With consideration of machine tool motion characteristics, an adaptive tool path generation is investigated, including establishing the functional relationship between spiral radius and revolution number with respect to cutting residual, smooth curve fitting and solving cutter contact points in the cutting direction with the consideration of interpolation error and tool radius compensation.A theoretical investigation for ATPG is conducted. The results indicate that the smooth curve produced by the spiral radius and revolution number can effectively suppress vibration of side-feeding motion, which can make the machining operated at a higher spindle speed.A comparison investigation on surface generation in ATPG and DiffSys was experimentally conducted on typical freeform surfaces. By adopting ATPG, the volume of tool path control points and machining time are effectively reduced, while surface form error as well as roughness is maintained. Therefore, the proposed ATPG significantly increases the machining efficiency for large aperture freeform optics.This research proposes a novel adaptive tool path generation (ATPG) for freeform optics to improve machining efficiency. The main conclusions are as follows: